The development and application of anti-tumor vaccines represents, perhaps one of the most challenging and important areas of research in tumor immunology today. Previous attempts at designing effective tumor vaccine protocols have yielded equivocal results, based in large part on the inability to define specific, tumor-associated antigens relevant to the promotion of a protective anti-tumor immune response and the inability to target vaccines towards the relevant arm(s) of the anti- tumor immune response. In the melanoma tumor system, it appears clear that cellular immunity is naturally generated in response to tumors since spontaneous regression of melanomas have been reported in a minority of patients. In addition, tumor-infiltrating lymphocytes (TIL) have been identified in most melanoma patients which display potent anti-melanoma cytolytic activity in vitro and in vivo. This cytolytic activity is mediated by CD8+ T cells which recognize melanoma-associated antigens in association with a major histocompatibility complex class I-restricting element. These melanoma antigens appear to represent peptides of limited heterogeneity that are shared by melanoma tumors bearing a common class I allele but derived from unrelated patients. The shared nature of such immune-reactive peptides makes them ideal candidates for development of anti-melanoma vaccines in humans. We propose to identify, isolate, and sequence melanoma peptides recognized by tumor-specific, HLA-A2 restricted TIL T cells. In initial experiments we have identified multiple melanoma peptides that are recognized by an oligoclonal CD8+ TIL line restricted by the HLA-A2 class I molecule. Further, we propose to examine the potential of such peptides (purified, synthetic, or synthetic variants) to promote or enhance a T cell-mediated immune response to melanoma tumor in vitro. The results of these studies will form the basis for novel anti-melanoma vaccine development in cases where CD8+ T cell reactivity against autologous tumor can be demonstrated, but where antigen specificity remains to be identified. Such an approach obviates the initial requirement to purify potentially immunogenic tumor- associated macromolecules or to identify and clone the genes encoding such proteins. Instead, this approach provides a mechanism in which full-processed and functionally-presented peptide fragments of these melanoma proteins are identified and characterized for their immunogenic potential to stimulate anti-tumor cytotoxic T cell responses in vitro and ultimate, in vivo. Our specific aims include: I. To identify and characterize melanoma peptides recognized by cytotoxic T cells from melanoma patients. II. To assess the immunogenic potential of melanoma-derived peptides presented by HLA-A2 class I molecules.